The division cycle of eukaryotic cells is regulated by a family of protein kinases known as the cyclin-dependent kinases (CDKs). The sequential activation of individual members of this family and their consequent phosphorylation of critical substrates promotes orderly progression through the cell cycle. In its simplest active form, a CDK consists of a catalytic subunit (the cyclin-dependent kinase) and a positive regulatory subunit known as a cyclin. In mammalian cells, the CDK family consists of at least seven members (Meyerson et al., (1992) EMBO J. 11:2909-2917; Xiong et al., (1992) Cell 71:505-514; Matsushime et al., (1992) Cell 71:323-334; Meyerson and Harlow, (1994) Mol. Cell Biol. 14:2077-2086; Fisher and Morgan, (1994) Cell 78:713-724). Combination with an equally diverse family of cyclins yields numerous cell cycle regulatory enzymes each with a potentially unique function. In mammals, more than one kinase subunit is implicated in cell-cycle control. For example, progression from G1 to S phase involves CdK4/cyclin E; S phase Cdk2/cyclin A; and M phase Cdc2/cyclin B (Nasmyth and Hunt, (1993) Nature 366:634-635).
The activity of CDKs is controlled by several mechanisms that include stimulatory and inhibitory phosphorylation events, and complex formation with other proteins. To become active, a CDKs require the association with its corresponding cyclin. Many cyclins either oscillate in abundance during the cell cycle or require the presence of growth factors for expression (see, for example, Nigg, (1993) Trends Cell Biol. 3:296; Sherr, (1993) Cell 73:1059-1065). For example, human CDK4 exclusively associates with the D-type cyclins (D1, D2, and D3) (Xiong et al., (1992) supra; Xiong et al., (1993a) Genes and Development 7:1572-1583; and Matsushime et al., (1991) Cell 65:701). The complexes formed by CDK4 and the D-type cyclins have been strongly implicated in the control of cell proliferation during the G1 phase (Motokura et al., (1993) Biochem. Biophys. Acta. 1155:63-78; Sherr, (1993) supra; Matsushimi et al., (1992) supra; and Kamb et al., (1994) Science 264:436-440). Once formed, cyclin/CDK complexes still require phosphorylation of a threonine residue (usually near amino acid 160) for activity (Solomon et al., (1993) EMBO J. 12:3133-3142; Fisher and Morgan, (1994) supra; Makela et al., (1994) Nature 371:254-257). Active enzymes can be further regulated by inhibitory phosphorylation (e.g. of Thr14 and Tyr15 in cdc2) (see Solomon, (1993) Curr. Opinion Cell Biol. 5:180-186; Dunphy, (1994) Trends Cell Biol. 4:202-207 for review) and by the binding of inhibitory proteins (e.g. p21 or p16) (reviewed in Hunter and Pines, (1994) Cell 79:573-582; Xiong et al., (1993b) Nature 366:701-704; Harper et al, (1993) Cell 75:805-816; Serrano et al., (1993) Nature 366:704-707).
Differences in the abilities of normal and transformed cells to proliferate had long predicted that alterations in the pathways that control cell cycle progression must accompany cellular transformation. Alteration in growth control pathways can translate into changes in the cell-cycle regulatory machinery, but the mechanisms by which this occurs are still poorly understood. This prompted a comparison of cyclin/CDK complexes present in normal fibroblasts to those present in their transformed derivatives (Xiong et al., (1993a) supra). In normal cells, each of the cyclin-dependent kinases exists in part in a quaternary complex consisting of a cyclin, a CDK, the proliferating cell nuclear antigen (PCNA) and the inhibitory protein p21 (Zhang et al., (1993) Mol. Biol. Cell 4:897-906). Quaternary complexes are lost from many transformed cells due to the absence of the tumor suppressor, p53, which directly controls the expression of p21 (Xiong et al., (1993a) supra; El-Deiry et al., (1993) Cell 75:817-825). In these transformed cells, p16 becomes the predominant partner of CDK4 and CDK6.
Cyclin A-associated enzymes have been established as key promoters of progression through the S-phase of the cell cycle (see Heichman and Roberts, (1994) Cell 79:557-562 for review). The expression of cyclin A and the activity of cyclin A/CDK2 kinase peaks during late G1 and S phase in both normal and transformed cells (Pines and Hunter, (1990) Nature 346:760-763). Furthermore, microinjection of antibodies to cyclin A or introduction of antisense cyclin A expression constructs can prevent DNA replication (Girard et al., (1991) Cell 67:1169-1179; Pagano et al., (1992) EMBO J.11:961-971). In some cell types, cyclin A has even been shown to localize to sites of ongoing DNA synthesis (Cardoso et al., (1993) Cell 74:979-992). Finally, ectopic expression of cyclin A was sufficient to allow adhesion independent DNA synthesis in normal rat kidney cells (Guadagno et al., (1993) Science 262:1572-1575).
Comparisons of cyclin kinase complexes in normal and transformed fibroblasts have identified a class of subunit rearrangements that specifically affected cyclin A/CDK2 complexes and replace the quaternary cyclin A/CDK2/p21/PCNA complexes found in normal cells (Xiong et al., (1993a) supra). The determination of the identities and biological functions of the subunits present in transformed cells that replace the quaternary cyclin A/CDK2/p21/PCNA complexes is highly desirable particularly in developing novel diagnostic and therapeutic applications to treat diseases characterized by deregulated cell growth and division.